Molecular mechanism of centrosome separation in budding yeast meiosis often referred to as the spindle pole body (SPB), the budding yeast centrosome shares structural components with, and is functionally equivalent to, the animal centrosome. Like centrosomes in human cells, duplicated SPBs in yeast are tethered by a proteinaceous structure, called the half-bridge, but how this linkage is maintained and then dissolved in a timely manner for spindle assembly is unclear at the molecular level. The objective of this project is to use the unique SPB cycle in budding yeast meiosis as a model to determine the regulation of centrosome cohesion and separation. During meiosis, duplicated SPBs are tethered for an extended G2 phase when recombination occurs; in addition, SPBs reduplicate before meiosis II, which is uncoupled from DNA replication. The process of SPB cohesion and separation is thus a key regulatory step during cell division. Previous works indicate that cleavage of the half-bridge likely permits SPB separation. Our laboratory recently demonstrated that posttranslational modification of a half-bridge subunit is required for proper SPB separation, and that a meiosis-specific protein protects SPB cohesion. We therefore hypothesize that the half-bridge subunit we identified is the site of protein cleavage, which is regulated by posttranslational modification and by the meiotic guardian protein. Three specific aims will be fulfilled to test this hypothesis: We will determine (1) how half-bridge proteins establish SPB cohesion, (2) how SPB cohesion is maintained during the G2 phase, and (3) how SPB cohesion is dissolved for spindle assembly. Under the first aim, the protein-interaction network at the half-bridge that mediates SPB cohesion will be determined. Under the second aim, the guardian protein that protects SPB cohesion will be characterized. Under the third aim, a meiotic SPB phosphoproteome map, generated in the applicant's lab, will guide the study of protein phosphorylation required for SPB separation. The proposed research is expected to reveal the molecular mechanism of SPB cohesion and separation, which is coordinated with spindle assembly and chromosome segregation. Because three of the four half-bridge subunits in yeast have known homologs in human cells, the proposed work is expected to provide insights into the regulation of mammalian centrosome separation and thereby contribute to elucidating the causes of chromosome missegregation and aneuploidy in humans.